PROJECT SUMMARY Regulatory T cells (Treg) play a crucial role in keeping the immune system in balance and preventing autoimmune disease. Defective Treg function leads to autoimmune diseases including rheumatoid arthritis and multiple sclerosis. On the other hand, Treg cells inside tumors can block effective anti-tumor immune responses. The development and maintenance of the Treg cell lineage are dependent on the transcription factor Foxp3, as loss of function mutations lead to severe lymphoproliferative disease in mice and humans. Thus, understanding the mechanisms that govern Foxp3 induction and stability may lead to the development of novel therapies for autoimmune disease and cancer. Dr. Zheng and colleagues recently developed a system to perform genome- wide CRISPR/Cas9 knockout screens to identify Foxp3 regulators in mouse induced and natural Tregs. The unbiased screen results not only confirmed a number of known Foxp3 regulators but also revealed many novel factors that control Foxp3 expression. Gene ontology analysis of the newly identified Foxp3 regulators revealed significant enrichment of multiple genes encoding subunits of the BAF (SWI/SNF) chromatin remodeling complexes, indicating an unknown role for this regulator in Treg development. Among the three BAF related complexes, the newly described GBAF complex promotes the expression of Foxp3, whereas the PBAF complex represses its expression. Furthermore, deletion of the bromodomain-containing protein Brd9, specific to GBAF complexes, led to reduced Foxp3 expression and compromised Treg function. Additionally, bromodomain inhibitors and chemical degraders of BRD9 act similarly to Brd9 genetic deletion to impair Foxp3 expression and Treg suppressor function. RNA-seq and ChIP-seq studies suggest that Brd9 cooperates with Foxp3 to potentiate its binding to Foxp3 target genes and regulate their expression. The overall objective of this study is to define the role of BAF related complexes in Treg function through regulation of and cooperation with Foxp3. This goal will be accomplished by elucidating the role of G/PBAF in Foxp3 induction and maintenance (Aim 1), defining the biochemical association of G/PBAF complexes with Foxp3 (Aim 2), identifying the functional role of GBAF in Foxp3-dependent transcription (Aim 3), and characterizing the functional consequences of disrupting G/PBAF subunits in Tregs in autoimmune disease and cancer models (Aim 4). The outcomes of the proposed studies are expected to fundamentally advance the understanding of epigenetic control of Foxp3 gene expression and Treg transcriptional networks. Additionally, these studies will provide key insights into the function of the newly described GBAF complex and its relationship with PBAF complexes. Finally, the outcomes of this research could provide evidence to support using drugs to target BAF related complexes for the treatment of autoimmune diseases and cancer.